Biological products

ABSTRACT

There is disclosed antibody molecules containing at least one CDR derived from a mouse monoclonal antibody having specificity for human CD22. There is also disclosed a CDR grafted antibody wherein at least one of the CDRs is a modified CDR. Further disclosed are DNA sequences encoding the chains of the antibody molecules, vectors, transformed host cells and uses of the antibody molecules in the treatment of diseases mediated by cells expressing CD22.

[0001] This application claims priority under 35 U.S.C. §119(a)-(d) toUnited Kingdom Application No. GB 0210121.0, filed May 2, 2002, and theapplication hereby is incorporated by reference in its entirety.

[0002] The present invention relates to an antibody molecule havingspecificity for antigenic determinants of the B lymphocyte antigen,CD22. The present invention also relates to the therapeutic uses of theantibody molecule and methods for producing the antibody molecule.

[0003] In a natural antibody molecule, there are two heavy chains andtwo light chains. Each heavy chain and each light chain has at itsN-terminal end a variable domain. Each variable domain is composed offour framework regions (FRs) alternating with three complementaritydetermining regions (CDRs). The residues in the variable domains areconventionally numbered according to a system devised by Kabat et al.This system is set forth in Kabat et al, 1987, in Sequences of Proteinsof Immunological Interest, US Department of Health and Human Services,NIH, USA (hereafter “Kabat et al. (supra)”). This numbering system isused in the present specification except where otherwise indicated.

[0004] The Kabat residue designations do not always correspond directlywith the linear numbering of the amino acid residues. The actual linearamino acid sequence may contain fewer or additional amino acids than inthe strict Kabat numbering corresponding to a shortening of, orinsertion into, a structural component, whether framework or CDR, of thebasic variable domain structure. The correct Kabat numbering of residuesmay be determined for a given antibody by alignment of residues ofhomology in the sequence of the antibody with a “standard” Kabatnumbered sequence.

[0005] The CDRs of the heavy chain variable domain are located atresidues 31-35 (CDR-H1), residues 50-65 (CDR-H2) and residues 95-102(CDR-H3) according to the Kabat numbering.

[0006] The CDRs of the light chain variable domain are located atresidues 24-34 (CDR-L1), residues 50-56 (CDR-L2) and residues 89-97(CDR-L3) according to the Kabat numbering.

[0007] Construction of CDR-grafted antibodies is described in EuropeanPatent Application EP-A-0239400, which discloses a process in which theCDRs of a mouse monoclonal antibody are grafted onto the frameworkregions of the variable domains of a human immunoglobulin by sitedirected mutagenesis using long oligonucleotides. The CDRs determine theantigen binding specificity of antibodies and are relatively shortpeptide sequences carried on the framework regions of the variabledomains.

[0008] The earliest work on humanising monoclonal antibodies byCDR-grafting was carried out on monoclonal antibodies recognisingsynthetic antigens, such as NP. However, examples in which a mousemonoclonal antibody recognising lysozyme and a rat monoclonal antibodyrecognising an antigen on human T-cells were humanised by CDR-graftinghave been described by Verhoeyen et al. (Science, 239, 1534-1536, 1988)and Riechmann et al. (Nature, 332, 323-324, 1988), respectively.

[0009] Riechmann et al., found that the transfer of the CDRs alone (asdefined by Kabat (Kabat et al. (supra) and Wu et al., J. Exp. Med., 132,211-250, 1970)) was not sufficient to provide satisfactory antigenbinding activity in the CDR-grafted product. It was found that a numberof framework residues have to be altered so that they correspond tothose of the donor framework region. Proposed criteria for selectingwhich framework residues need to be altered are described inInternational Patent Application No. WO 90/07861.

[0010] A number of reviews discussing CDR-grafted antibodies have beenpublished, including Vaughan et al. (Nature Biotechnology, 16, 535-539,1998).

[0011] Malignant lymphomas are a diverse group of neoplasms. Themajority of cases occur in older people. Non-Hodgkins Lymphoma (NHL) isa disease that currently affects 200,000 to 250,000 patients in the U.S.It is the second fastest rising cancer in the U.S., rising at a rate ofabout 55,000 new cases per year. The incidence is rising at a rate thatis greater than can be accounted for simply by the increasing age of thepopulation and exposure to known risk factors.

[0012] The classification of lymphoma is complex, and has evolved inrecent decades. In 1994 the Revised European-American Lymphoma (REAL)classification was introduced. This classification organises lymphomasof B cell (the most frequently identified), T cell and unclassifiableorigin into agreed subtypes. In everyday practice, the grouping of NHLsinto low, intermediate and high-grade categories on the basis of theirgeneral histological appearance, broadly reflects their clinicalbehaviour.

[0013] NHL predominantly affects the lymph nodes but, in individualpatients, the tumour may involve other anatomical sites such as theliver, spleen, bone marrow, lung, gut and skin. The disease commonlypresents as a painless enlargement of lymph nodes. Extranodal lymphomamost frequently affects the gut, although primary lymphoma of virtuallyevery organ has been documented. Systemic symptoms include fever,sweats, tiredness and weight loss.

[0014] Until recently, the Ann Arbor staging system, based entirely uponthe anatomical extent of disease, was the major determinant of therapyin NHL. This information may be refined by incorporating additionalprognostic pointers, including age, serum lactate dehydrogenase levelsand performance status. Even so, knowledge of the Ann Arbor stagingsystem, together with the histological and immunological subtype of thetumour, is still the major determinant of treatment.

[0015] Low grade NHL has an indolent course, with a median patientsurvival of 8 to 10 years. Survival is little impacted by currentlyavailable therapy, although irradiation of local disease andchemotherapy for systemic symptoms improves patients' quality of life.Combination chemotherapy may be reserved for relapsed disease.Intermediate disease and, especially, high grade disease is extremelyaggressive and tends to disseminate. Disease of this grade requiresurgent treatment. Radiotherapy may be a useful component of treatment inpatients with very bulky disease. Many different chemotherapy regimenshave been employed, and long-term disease-free survival may be obtainedin more than half of patients. High dose therapy with stem cell supportwas introduced initially for patients with relapsed or refractorydisease, but is now increasingly finding a place in first line therapyfor patients with poor-risk disease. The tendency in recent years for anincreasingly aggressive therapeutic approach must be balanced againstthe generally elderly age and relative debility of many patients withNHL, and by the need to match the toxicity of treatment to theindividual prognosis of each patient's disease.

[0016] Improved treatments, that are more effective and bettertolerated, are needed. Agents recently introduced include new cytotoxicdrugs, progressively incorporated into combinations, and theintroduction of antibody-based therapies.

[0017] Non-Hodgkin's lymphoma encompasses a range of B cell lymphomas. Bcell antigens therefore represent suitable targets for antibody therapy.

[0018] CD22 is a 135 kDa membrane glycoprotein belonging to a family ofsialic acid binding proteins called sialoadhesins. It is detected in thecytoplasm early in B cell development, appears on the cell surfacesimultaneously with IgD and is found on most mature B cells. Expressionis increased following B cell activation. CD22 is lost with terminaldifferentiation and is generally reported as being absent on plasmacells. Thus this internalising antigen is present on the surface ofpre-B cells and mature B cells but not stem cells or plasma cells.

[0019] Two isoforms of CD22 exist in man. The predominant form (CD22β)contains 7 immunoglobulin-like (Ig-like) domains in the extracellularregion. The CD22α variant lacks Ig-like domain 4 and may have atruncated cytoplasmic domain. Antibodies which block CD22 adhesion tomonocytes, neutrophils, lymphocytes and erythrocytes have been shown tobind within the first or second Ig-like domain.

[0020] The cytoplasmic domain of CD22 is tyrosine phosphorylated uponligation of the B cell antigen receptor and associates with Lyk, Syk andphosphatidyl inositol 3-kinase. The function of CD22 is to down-modulatethe B cell activation threshold. It can also mediate cell adhesionthrough interaction with cells bearing the appropriatesialoglycoconjugates.

[0021] CD22 is expressed in most B cell leukaemias and lymphomas,including NHL, acute lymphoblastic leukaemia (B-ALL), chroniclymphocytic leukaemia (B-CLL) and especially acute non-lymphocyticleukaemia (ANLL).

[0022] Monoclonal antibodies against CD22 have been described in theprior art. WO 98/41641 describes recombinant anti-CD22 antibodies withcysteine residues at V_(H)44 and V_(L)100. WO 96/04925 describes theV_(H) and V_(L) regions of the anti-CD22 antibody LL2. U.S. Pat. No.5,686,072 describes combinations of anti-CD22 and anti-CD19immunotoxins. WO 98/42378 describes the use of naked anti-CD22antibodies for the treatment of B-cell malignancies.

[0023] A number of antibody-based therapeutics have either been recentlylicensed, e.g. Rituxan (an unlabelled chimeric human γ1 (+mγ1V-region)specific for CD20), or are in clinical trials for this disease. Theserely either on complement- or ADCC- mediated killing of B cells or theuse of radionuclides, such as ¹³¹I or ⁹⁰Y, which have associatedpreparation and use problems for clinicians and patients. There is aneed for an antibody molecule to treat NHL which can be used repeatedlyand produced easily and efficiently. There is also a need for anantibody molecule, which has high affinity for CD22 and lowimmunogenicity in humans.

SUMMARY OF THE INVENTION

[0024] In a first aspect, the present invention provides an antibodymolecule having specificity for human CD22, comprising a heavy chainwherein the variable domain comprises a CDR (as defined by Kabat et al.,(supra)) having the sequence given as H1 in FIG. 1 (SEQ ID NO:1) forCDR-H1, as H2 in FIG. 1 (SEQ ID NO:2) or an H2 from which a potentialglycosylation site has been removed, or an H2 in which the lysineresidue at position 60 (according to the Kabat numbering system) hasbeen replaced by an alternative amino acid, or an H2 in which both theglycosylation site and the reactive lysine at position 60 have beenremoved for CDR-H2 or as H3 in FIG. 1 (SEQ ID NO:3) for CDR-H3.

[0025] The antibody molecule of the first aspect of the presentinvention comprises at least one CDR selected from H1, H2 and H3 (SEQ IDNO:1, SEQ ID NO:2 and SEQ ID NO:3) for the heavy chain variable domain.Preferably, the antibody molecule comprises at least two and morepreferably all three CDRs in the heavy chain variable domain.

[0026] In a second aspect of the present invention, there is provided anantibody molecule having specificity for human CD22, comprising a lightchain wherein the variable domain comprises a CDR (as defined by Kabatet al., (supra)) having the sequence given as L1 in FIG. 1 (SEQ ID NO:4)for CDR-L1, L2 in FIG. 1 (SEQ ID NO:5) for CDR-L2 or L3 in FIG. 1 (SEQID NO:6) for CDR-L3.

[0027] The antibody molecule of the second aspect of the presentinvention comprises at least one CDR selected from L1, L2 and L3 (SEQ IDNO:4; SEQ ID NO:5 and SEQ ID NO:6) for the light chain variable domain.Preferably, the antibody molecule comprises at least two and morepreferably all three CDRs in the light chain variable domain.

[0028] The antibody molecules of the first and second aspects of thepresent invention preferably have a complementary light chain or acomplementary heavy chain, respectively.

[0029] Preferably, the antibody molecule of the first or second aspectof the present invention comprises a heavy chain wherein the variabledomain comprises a CDR (as defined by Kabat et al., (supra)) having thesequence given as HI in FIG. 1 (SEQ ID NO:1) for CDR-H1, as H2 in FIG. 1(SEQ ID NO:2) or an H2 from which a potential glycosylation site hasbeen removed, or an H2 in which the lysine residue at position 60(according to the Kabat numbering system) has been replaced by analternative amino acid, or an H2 in which both the glycosylation siteand the reactive lysine at position 60 have been removed for CDR-H2 oras H3 in FIG. 1 (SEQ ID NO:3) for CDR-H3 and a light chain wherein thevariable domain comprises a CDR (as defined by Kabat et al., (supra))having the sequence given as L1 in FIG. 1 (SEQ ID NO:4) for CDR-L1, asL2 in FIG. 1 (SEQ ID NO:5) for CDR-L2 or as L3 in FIG. 1 (SEQ ID NO:6)for CDR-L3.

[0030] The CDRs given in SEQ IDS NOS:1 to 6 and in FIG. 1 referred toabove are derived from a mouse monoclonal antibody 5/44.

[0031] The complete sequences of the variable domains of the mouse 5/44antibody are shown in FIG. 2 (light chain) (SEQ ID NO:7) and FIG. 3(heavy chain) (SEQ ID NO:8). This mouse antibody is also referred tobelow as “the donor antibody” or the “murine monoclonal antibody”.

[0032] A first alternatively preferred embodiment of the first or secondaspect of the present invention is the mouse monoclonal antibody 5/44having the light and heavy chain variable domain sequences shown in FIG.2 (SEQ ID NO:7) and FIG. 3 (SEQ ID NO:8), respectively. The light chainconstant region of 5/44 is kappa and the heavy chain constant region isIgG1.

[0033] In a second alternatively preferred embodiment, the antibodyaccording to either of the first and second aspects of the presentinvention is a chimeric mouse/human antibody molecule, referred toherein as the chimeric 5/44 antibody molecule. The chimeric antibodymolecule comprises the variable domains of the mouse monoclonal antibody5/44 (SEQ ID NOS:7 and 8) and human constant domains. Preferably, thechimeric 5/44 antibody molecule comprises the human C kappa domain(Hieter et al., Cell, 22, 197-207, 1980; Genebank accession numberJ00241) in the light chain and the human gamma 4 domains (Flanagan etal., Nature, 300, 709-713, 1982) in the heavy chain, optionally with theserine residue at position 241 replaced by a proline residue.

[0034] Preferably, the antibody of the present invention comprises aheavy chain wherein the variable domain comprises as CDR-H2 (as definedby Kabat et al., (supra)) an H2′ in which a potential glycosylation sitesequence has been removed and which unexpectedly increased the affinityof the chimeric 5/44 antibody for the CD22 antigen and which preferablyhas as CDR-H2 the sequence given as H2′ (SEQ ID NO: 13).

[0035] Alternatively or additionally, the antibody of the presentinvention may comprise a heavy chain wherein the variable domaincomprises as CDR-H2 (as defined by Kabat et al., (supra)) an H2″ inwhich a lysine residue at position 60, which is located at an exposedposition within CDR-H2 and which is considered to have the potential toreact with conjugation agents resulting in a reduction of antigenbinding affinity, is substituted for an alternative amino acid to resultin a conserved substitution. Preferably CDR-H2 has the sequence given asH2″ (SEQ ID NO:15).

[0036] Alternatively or additionally, the antibody of the presentinvention may comprise a heavy chain wherein the variable domaincomprises as CDR-H2 (as defined by Kabat et al., (supra)) an H2′″ inwhich both the potential glycosylation site sequence and the lysineresidue at position 60, are substituted for alternative amino acids.Preferably CDR-H2 has the sequence given as H2′″ (SEQ ID NO: 16).

[0037] In a third alternatively preferred embodiment, the antibodyaccording to either of the first and second aspects of the presentinvention is a CDR-grafted antibody molecule. The term “a CDR-graftedantibody molecule” as used herein refers to an antibody molecule whereinthe heavy and/or light chain contains one or more CDRs (including, ifdesired, a modified CDR) from a donor antibody (e.g. a murine monoclonalantibody) grafted into a heavy and/or light chain variable regionframework of an acceptor antibody (e.g. a human antibody).

[0038] Preferably, such a CDR-grafted antibody has a variable domaincomprising human acceptor framework regions as well as one or more ofthe donor CDRs referred to above.

[0039] When the CDRs are grafted, any appropriate acceptor variableregion framework sequence may be used having regard to the class/type ofthe donor antibody from which the CDRs are derived, including mouse,primate and human framework regions. Examples of human frameworks whichcan be used in the present invention are KOL, NEWM, REI, EU, TUR, TEI,LAY and POM (Kabat et al. (supra)). For example, KOL and NEWM can beused for the heavy chain, REI can be used for the light chain and EU,LAY and POM can be used for both the heavy chain and the light chain.Alternatively, human germline sequences may be used. The preferredframework region for the light chain is the human germline sub-groupsequence (DPK9+JK1) shown in FIG. 5 (SEQ ID NO:17). The preferredframework region for the heavy chain is the human sub-group sequence(DP7+JH4) shown in FIG. 6 (SEQ ID NO:21).

[0040] In a CDR-grafted antibody of the present invention, it ispreferred to use as the acceptor antibody one having chains which arehomologous to the chains of the donor antibody. The acceptor heavy andlight chains do not necessarily need to be derived from the sameantibody and may, if desired, comprise composite chains having frameworkregions derived from different chains.

[0041] Also, in a CDR-grafted antibody of the present invention, theframework regions need not have exactly the same sequence as those ofthe acceptor antibody. For instance, unusual residues may be changed tomore frequently-occurring residues for that acceptor chain class ortype. Alternatively, selected residues in the acceptor framework regionsmay be changed so that they correspond to the residue found at the sameposition in the donor antibody or to a residue that is a conservativesubstitution for the residue found at the same position in the donorantibody. Such changes should be kept to the minimum necessary torecover the affinity of the donor antibody. A protocol for selectingresidues in the acceptor framework regions which may need to be changedis set forth in WO 91/09967.

[0042] Preferably, in a CDR-grafted antibody molecule according to thepresent invention, if the acceptor light chain has the human sub-groupDPK9+JK1 sequence (shown in FIG. 2) (SEQ ID NO:17) then the acceptorframework regions of the light chain comprise donor residues atpositions 2, 4, 37, 38, 45 and 60 and may additionally comprise a donorresidue at position 3 (according to Kabat et al. (supra)).

[0043] Preferably, in a CDR-grafted antibody molecule of the presentinvention, if the acceptor heavy chain has the human DP7+JH4 sequence(shown in FIG. 3) (SEQ ID NO:21), then the acceptor framework regions ofthe heavy chain comprise, in addition to one or more donor CDRs, donorresidues at positions 1, 28, 48, 71 and 93 and may additionally comprisedonor residues at positions 67 and 69 (according to Kabat et al.(supra)).

[0044] Donor residues are residues from the donor antibody, i.e. theantibody from which the CDRs were originally derived.

[0045] Preferably, the antibody of the present invention comprises aheavy chain wherein the variable domain comprises as CDR-H2 (as definedby Kabat et al., (supra)) an H2′ in which a potential glycosylation sitesequence has been removed in order to increase the affinity of thechimeric 5/44 antibody for the CD22 antigen and which preferably has asCDR-H2 the sequence given as H2′ (SEQ ID NO: 13).

[0046] Alternatively or additionally, the antibody of the presentinvention may comprise a heavy chain wherein the variable domaincomprises as CDR-H2 (as defined by Kabat et al., (supra)) an H2″ inwhich a lysine residue at position 60, which is located at an exposedposition within CDR-H2 and which is considered to have the potential toreact with conjugation agents resulting in a reduction of antigenbinding affinity, is substituted for an alternative amino acid.Preferably CDR-H2 has the sequence given as H2″ (SEQ ID NO:15).

[0047] Alternatively or additionally, the antibody of the presentinvention may comprise a heavy chain wherein the variable domaincomprises as CDR-H2 (as defined by Kabat et al., (supra)) an H2′″ inwhich both the potential glycosylation site sequence and the lysineresidue at position 60, are substituted for alternative amino acids.Preferably CDR-H2 has the sequence given as H2′″ (SEQ ID NO:16).

[0048] The antibody molecule of the present invention may comprise: acomplete antibody molecule, having full length heavy and light chains; afragment thereof, such as a Fab, modified Fab, Fab′, F(ab′)₂ or Fvfragment; a light chain or heavy chain monomer or dimer; a single chainantibody, e.g. a single chain Fv in which the heavy and light chainvariable domains are joined by a peptide linker. Similarly, the heavyand light chain variable regions may be combined with other antibodydomains as appropriate.

[0049] The antibody molecule of the present invention may have aneffector or a reporter molecule attached to it. For instance, it mayhave a macrocycle, for chelating a heavy metal atom, or a toxin, such asricin, attached to it by a covalent bridging structure. Alternatively,procedures of recombinant DNA technology may be used to produce anantibody molecule in which the Fc fragment (CH2, CH3 and hinge domains),the CH2 and CH3 domains or the CH3 domain of a complete immunoglobulinmolecule has (have) been replaced by, or has (have) attached thereto bypeptide linkage, a functional non-immunoglobulin protein, such as anenzyme or toxin molecule.

[0050] The antibody molecule of the present invention preferably has abinding affinity of at least 0.85×10⁻¹⁰ M, more preferably at least0.75×10⁻¹⁰ M and most preferably at least 0.5×10⁻¹⁰ M.

[0051] Preferably, the antibody molecule of the present inventioncomprises the light chain variable domain 5/44-gL1 (SEQ ID NO: 19) andthe heavy chain variable domain 5/44-gH7 (SEQ ID NO:27). The sequencesof the variable domains of these light and heavy chains are shown inFIGS. 5 and 6, respectively.

[0052] The present invention also relates to variants of the antibodymolecule of the present invention, which have an improved affinity forCD22. Such variants can be obtained by a number of affinity maturationprotocols including mutating the CDRs (Yang et al., J. Mol. Biol., 254,392-403, 1995), chain shuffling (Marks et al., Bio/Technology, 10,779-783, 1992), use of mutator strains of E. coli (Low et al., J. Mol.Biol., 250, 359-368, 1996), DNA shuffling (Patten et al., Curr. Opin.Biotechnol., 8, 724-733, 1997), phage display (Thompson et al., J. Mol.Biol., 256, 77-88, 1996) and sexual PCR (Crameri et al., Nature, 391,288-291, 1998). Vaughan et al. (supra) discusses these methods ofaffinity maturation.

[0053] The present invention also provides a DNA sequence encoding theheavy and/or light chain(s) of the antibody molecule of the presentinvention.

[0054] Preferably, the DNA sequence encodes the heavy or the light chainof the antibody molecule of the present invention.

[0055] The DNA sequence of the present invention may comprise syntheticDNA, for instance produced by chemical processing, cDNA, genomic DNA orany combination thereof.

[0056] The present invention also relates to a cloning or expressionvector comprising one or more DNA sequences of the present invention.Preferably, the cloning or expression vector comprises two DNAsequences, encoding the light chain and the heavy chain of the antibodymolecule of the present invention, respectively.

[0057] General methods by which the vectors may be constructed,transfection methods and culture methods are well known to those skilledin the art. In this respect, reference is made to “Current Protocols inMolecular Biology”, 1999, F. M. Ausubel (ed), Wiley Interscience, NewYork and the Maniatis Manual produced by Cold Spring Harbor Publishing.

[0058] DNA sequences which encode the antibody molecule of the presentinvention can be obtained by methods well known to those skilled in theart. For example, DNA sequences coding for part or all of the antibodyheavy and light chains may be synthesised as desired from the determinedDNA sequences or on the basis of the corresponding amino acid sequences.

[0059] DNA coding for acceptor framework sequences is widely availableto those skilled in the art and can be readily synthesised on the basisof their known amino acid sequences.

[0060] Standard techniques of molecular biology may be used to prepareDNA sequences coding for the antibody molecule of the present invention.Desired DNA sequences may be synthesised completely or in part usingoligonucleotide synthesis techniques. Site-directed mutagenesis andpolymerase chain reaction (PCR) techniques may be used as appropriate.

[0061] Any suitable host cell/vector system may be used for expressionof the DNA sequences encoding the antibody molecule of the presentinvention. Bacterial, for example E. coli, and other microbial systemsmay be used, in part, for expression of antibody fragments such as Faband F(ab′)₂ fragments, and especially Fv fragments and single chainantibody fragments, for example, single chain Fvs. Eukaryotic, e.g.mammalian, host cell expression systems may be used for production oflarger antibody molecules, including complete antibody molecules.Suitable mammalian host cells include CHO, myeloma or hybridoma cells.

[0062] The present invention also provides a process for the productionof an antibody molecule according to the present invention comprisingculturing a host cell containing a vector of the present invention underconditions suitable for leading to expression of protein from DNAencoding the antibody molecule of the present invention, and isolatingthe antibody molecule.

[0063] The antibody molecule may comprise only a heavy or light chainpolypeptide, in which case only a heavy chain or light chain polypeptidecoding sequence needs to be used to transfect the host cells. Forproduction of products comprising both heavy and light chains, the cellline may be transfected with two vectors, a first vector encoding alight chain polypeptide and a second vector encoding a heavy chainpolypeptide. Alternatively, a single vector may be used, the vectorincluding sequences encoding light chain and heavy chain polypeptides.

[0064] The present invention also provides a therapeutic or diagnosticcomposition comprising an antibody molecule of the present invention incombination with a pharmaceutically acceptable excipient, diluent orcarrier.

[0065] The present invention also provides a process for preparation ofa therapeutic or diagnostic composition comprising admixing the antibodymolecule of the present invention together with a pharmaceuticallyacceptable excipient, diluent or carrier.

[0066] The antibody molecule may be the sole active ingredient in thetherapeutic or diagnostic composition or may be accompanied by otheractive ingredients including other antibody ingredients, for exampleanti-T cell, anti-IFNγ or anti-LPS antibodies, or non-antibodyingredients such as xanthines.

[0067] The pharmaceutical compositions preferably comprise atherapeutically effective amount of the antibody of the invention. Theterm “therapeutically effective amount” as used herein refers to anamount of a therapeutic agent needed to treat, ameliorate or prevent atargeted disease or condition, or to exhibit a detectable therapeutic orpreventative effect. For any antibody, the therapeutically effectivedose can be estimated initially either in cell culture assays or inanimal models, usually in rodents, rabbits, dogs, pigs or primates. Theanimal model may also be used to determine the appropriate concentrationrange and route of administration. Such information can then be used todetermine useful doses and routes for administration in humans.

[0068] The precise effective amount for a human subject will depend uponthe severity of the disease state, the general health of the subject,the age, weight and gender of the subject, diet, time and frequency ofadministration, drug combination(s), reaction sensitivities andtolerance/response to therapy. This amount can be determined by routineexperimentation and is within the judgement of the clinician. Generally,an effective dose will be from 0.01 mg/kg to 50 mg/kg, preferably 0.1mg/kg to 20 mg/kg, more preferably about 15 mg/kg.

[0069] Compositions may be administered individually to a patient or maybe administered in combination with other agents, drugs or hormones.

[0070] The dose at which the antibody molecule of the present inventionis administered depends on the nature of the condition to be treated,the grade of the malignant lymphoma or leukaemia and on whether theantibody molecule is being used prophylactically or to treat an existingcondition.

[0071] The frequency of dose will depend on the half-life of theantibody molecule and the duration of its effect. If the antibodymolecule has a short half-life (e.g. 2 to 10 hours) it may be necessaryto give one or more doses per day. Alternatively, if the antibodymolecule has a long half life (e.g. 2 to 15 days) it may only benecessary to give a dosage once per day, once per week or even onceevery 1 or 2 months.

[0072] A pharmaceutical composition may also contain a pharmaceuticallyacceptable carrier for administration of the antibody. The carriershould not itself induce the production of antibodies harmful to theindividual receiving the composition and should not be toxic. Suitablecarriers may be large, slowly metabolised macromolecules such asproteins, polypeptides, liposomes, polysaccharides, polylactic acids,polyglycolic acids, polymeric amino acids, amino acid copolymers andinactive virus particles.

[0073] Pharmaceutically acceptable salts can be used, for examplemineral acid salts, such as hydrochlorides, hydrobromides, phosphatesand sulphates, or salts of organic acids, such as acetates, propionates,malonates and benzoates.

[0074] Pharmaceutically acceptable carriers in therapeutic compositionsmay additionally contain liquids such as water, saline, glycerol andethanol. Additionally, auxiliary substances, such as wetting oremulsifying agents or pH buffering substances, may be present in suchcompositions. Such carriers enable the pharmaceutical compositions to beformulated as tablets, pills, dragees, capsules, liquids, gels, syrups,slurries and suspensions, for ingestion by the patient.

[0075] Preferred forms for administration include forms suitable forparenteral administration, e.g. by injection or infusion, for example bybolus injection or continuous infusion. Where the product is forinjection or infusion, it may take the form of a suspension, solution oremulsion in an oily or aqueous vehicle and it may contain formulatoryagents, such as suspending, preservative, stabilising and/or dispersingagents. Alternatively, the antibody molecule may be in dry form, forreconstitution before use with an appropriate sterile liquid.

[0076] Once formulated, the compositions of the invention can beadministered directly to the subject. The subjects to be treated can beanimals. However, it is preferred that the compositions are adapted foradministration to human subjects.

[0077] The pharmaceutical compositions of this invention may beadministered by any number of routes including, but not limited to,oral, intravenous, intramuscular, intra-arterial, intramedullary,intrathecal, intraventricular, transdermal, transcutaneous (for example,see WO98/20734), subcutaneous, intraperitoneal, intranasal, enteral,topical, sublingual, intravaginal or rectal routes. Hyposprays may alsobe used to administer the pharmaceutical compositions of the invention.Typically, the therapeutic compositions may be prepared as injectables,either as liquid solutions or suspensions. Solid forms suitable forsolution in, or suspension in, liquid vehicles prior to injection mayalso be prepared.

[0078] Direct delivery of the compositions will generally beaccomplished by injection, subcutaneously, intraperitoneally,intravenously or intramuscularly, or delivered to the interstitial spaceof a tissue. The compositions can also be administered into a lesion.Dosage treatment may be a single dose schedule or a multiple doseschedule.

[0079] It will be appreciated that the active ingredient in thecomposition will be an antibody molecule. As such, it will besusceptible to degradation in the gastrointestinal tract. Thus, if thecomposition is to be administered by a route using the gastrointestinaltract, the composition will need to contain agents which protect theantibody from degradation but which release the antibody once it hasbeen absorbed from the gastrointestinal tract.

[0080] A thorough discussion of pharmaceutically acceptable carriers isavailable in Remington's Pharmaceutical Sciences (Mack PublishingCompany, N.J. 1991).

[0081] It is also envisaged that the antibody of the present inventionwill be administered by use of gene therapy. In order to achieve this,DNA sequences encoding the heavy and light chains of the antibodymolecule under the control of appropriate DNA components are introducedinto a patient such that the antibody chains are expressed from the DNAsequences and assembled in situ.

[0082] The present invention also provides the antibody molecule of thepresent invention for use in treating a disease mediated by cellsexpressing CD22.

[0083] The present invention further provides the use of the antibodymolecule according to the present invention in the manufacture of amedicament for the treatment of a disease mediated by cells expressingCD22.

[0084] The antibody molecule of the present invention may be utilised inany therapy where it is desired to reduce the level of cells expressingCD22 that are present in the human or animal body. These CD22-expressingcells may be circulating in the body or be present in an undesirablyhigh level localised at a particular site in the body. For example,elevated levels of cells expressing CD22 will be present in B celllymphomas and leukaemias. The antibody molecule of the present inventionmay be utilised in the therapy of diseases mediated by cells expressingCD22.

[0085] The antibody molecule of the present invention is preferably usedfor treatment of malignant lymphomas and leukaemias, most preferablyNHL.

[0086] The present invention also provides a method of treating human oranimal subjects suffering from or at risk of a disorder mediated bycells expressing CD22, the method comprising administering to thesubject an effective amount of the antibody molecule of the presentinvention.

[0087] The antibody molecule of the present invention may also be usedin diagnosis, for example in the in vivo diagnosis and imaging ofdisease states involving cells that express CD22.

[0088] The present invention is further described by way of illustrationonly in the following examples, which refer to the accompanying Figures,in which:

[0089]FIG. 1 shows the amino acid sequence of the CDRs of mousemonoclonal antibody 5/44 (SEQ ID NOS: 1 to 6);

[0090]FIG. 2 shows the complete sequence of the light chain variabledomain of mouse monoclonal antibody 5/44;

[0091]FIG. 3 shows the complete sequence of the heavy chain variabledomain of mouse monoclonal antibody 5/44;

[0092]FIG. 4 shows the strategy for removal of the glycosylation siteand reactive lysine in CDR-H2;

[0093]FIG. 5 shows the graft design for the 5/44 light chain sequence;

[0094]FIG. 6 shows the graft design for the 5/44 heavy chain sequence;

[0095]FIG. 7 shows the vectors pMRR14 and pMRR10.1;

[0096]FIG. 8 shows the Biacore assay results of the chimeric 5/44mutants;

[0097]FIG. 9 shows the oligonucleotides for 5/44 gH1 and gL1 geneassemblies;

[0098]FIG. 10 shows the intermediate vectors pCR2.1(544gH1) andpCR2.1(544gL1);

[0099]FIG. 11 shows the oligonucleotide cassettes used to make furthergrafts;

[0100]FIG. 12 shows the competition assay between fluorescently labelledmouse 5/44 antibody and grafted variants; and

[0101]FIG. 13 shows the full DNA and protein sequence of the graftedheavy and light chains.

DETAILED DESCRIPTION OF THE INVENTION Example 1: Generation of CandidateAntibodies

[0102] A panel of antibodies against CD22 were selected from hybridomasusing the following selection criteria: binding to Daudi cells,internalisation on Daudi cells, binding to peripheral blood mononuclearcells (PBMC), internalisation on PBMC, affinity (greater than 10⁻⁹ M),mouse γ1 and production rate. 5/44 was selected as the preferredantibody.

Example 2: Gene Cloning and Expression of a Chimeric 5/44 AntibodyMolecule

[0103] Preparation of 5/44 Hybridoma Cells and RNA Preparation therefromHybridoma 5/44 was generated by conventional hybridoma technologyfollowing immunisation of mice with human CD22 protein. RNA was preparedfrom 5/44 hybridoma cells using a RNEasy kit (Qiagen, Crawley, UK;Catalogue No. 74106). The RNA obtained was reverse transcribed to cDNA,as described below.

[0104] Distribution of CD22 on NHL Tumours

[0105] An immunohistochemistry study was undertaken to examine theincidence and distribution of staining using the 5/44 anti-CD22monoclonal antibodies. Control anti-CD20 and anti-CD79a antibodies wereincluded in the study to confirm B cell areas of tumours.

[0106] A total of 50 tumours were studied and these were categorised asfollows by using the Working Formulation and REAL classificationsystems:

[0107] 7 B lymphoblastic leukaemia/lymphoma (High/l)

[0108] 4 B-CLL/small lymphocytic lymphoma (Low/A)

[0109] 3 lymphoplasmacytoid/Immunocytoma (Low/A)

[0110] 1 Mantle cell (Int/F)

[0111] 14 Follicle center lymphoma (Low to Int/D)

[0112] 13 Diffuse large cell lymphoma (Int to High/G,H)

[0113] 6 Unclassifiable (K)

[0114] 2 T cell lymphomas

[0115] 40 B cell lymphomas were positive for CD22 antigen with the 5/44antibody at 0.1 μg/ml and a further 6 became positive when theconcentration was increased to 0.5 μg/ml. For the remaining 2 B celltumours that were negative at 0.1 μg/ml, there was insufficient tissueremaining to test at the higher concentration. However, parallel testingwith another Celltech anti-CD22 antibody 6/13, which gave strongerstaining than 5/44, resulted in all 48 B cell lymphomas stainingpositive for CD22.

[0116] Thus, it is possible to conclude that the CD22 antigen is widelyexpressed on B cell lymphomas and thus provides a suitable target forimmunotherapy in NHL.

[0117] PCR Cloning of 5/44 V_(H) and V_(L)

[0118] cDNA sequences coding for the variable domains of 5/44 heavy andlight chains were synthesised using reverse transcriptase to producesingle stranded cDNA copies of the mRNA present in the total RNA. Thiswas then used as the template for amplification of the murine V-regionsequences using specific oligonucleotide primers by the Polymerase ChainReaction (PCR).

[0119] a) cDNA Synthesis

[0120] cDNA was synthesised in a 20 μl reaction volume containing thefollowing reagents: 50 mM Tris-HCl pH 8.3, 75 mM KCl, 10 mMdithiothreitol, 3 mM MgCl₂, 0.5 mM each deoxyribonucleosidetriphosphate, 20 units RNAsin, 75 ng random hexanucleotide primer, 2 μg5/44 RNA and 200 units Moloney Murine Leukemia Virus reversetranscriptase. After incubation at 42° C. for 60 minutes, the reactionwas terminated by heating at 95° C. for 5 minutes.

[0121] b) PCR

[0122] Aliquots of the cDNA were subjected to PCR using combinations ofprimers specific for the heavy and light chains. Degenerate primer poolsdesigned to anneal with the conserved sequences of the signal peptidewere used as forward primers. These sequences all contain, in order, arestriction site (V_(L) SfuI; V_(H) HindIII) starting 7 nucleotides fromtheir 5′ ends, the sequence GCCGCCACC (SEQ ID NO:50), to allow optimaltranslation of the resulting mRNAs, an initiation codon and 20-30nucleotides based on the leader peptide sequences of known mouseantibodies (Kabat et al., Sequences of proteins of immunologicalinterest, 5^(th) Edition, 1991, U.S. Department of Health and HumanServices, Public Health Service, National Institutes of Health).

[0123] The 3′ primers are designed to span the framework 4 J-C junctionof the antibody and contain a restriction site for the enzyme BsiWI tofacilitate cloning of the V_(L) PCR fragment. The heavy chain 3′ primersare a mixture designed to span the J-C junction of the antibody. The 3′primer includes an ApaI restriction site to facilitate cloning. The 3′region of the primers contains a mixed sequence based on those found inknown mouse antibodies (Kabat et al., 1991, supra).

[0124] The combinations of primers described above enable the PCRproducts for V_(H) and V1 to be cloned directly into an appropriateexpression vector (see below) to produce chimeric (mouse-human) heavyand light chains and for these genes to be expressed in mammalian cellsto produce chimeric antibodies of the desired isotype.

[0125] Incubations (100 μl) for the PCR were set up as follows. Eachreaction contained 10 mM Tris-HCl pH 8.3, 1.5 mM MgCl₂, 50 mM KCl, 0.01%w/v gelatin, 0.25 mM each deoxyribonucleoside triphosphate, 10 pmoles 5′primer mix, 10 pmoles 3′ primer, 1 μl cDNA and 1 unit Taq polymerase.Reactions were incubated at 95° C. for 5 minutes and then cycled through94° C. for 1 minute, 55° C. for 1 minute and 72° C. for 1 minute. After30 cycles, aliquots of each reaction were analysed by electrophoresis onan agarose gel.

[0126] For the heavy chain V-region, an amplified DNA product was onlyobtained when a primer pool annealing within the start of framework Ireplaced the signal peptide primer pool. The fragments were cloned intoDNA sequencing vectors. The DNA sequence was determined and translatedto give a deduced amino acid sequence. This deduced sequence wasverified by reference to the N-terminal protein sequence determinedexperimentally. FIGS. 2 and 3 shows the DNA/protein sequence of themature light and heavy chain V-regions of mouse monoclonal 5/44respectively.

[0127] c) Molecular Cloning of the PCR Fragments

[0128] The murine v-region sequences were then cloned into theexpression vectors pMRR10.1 and pMRR14 (FIG. 7). These are vectors forthe expression of light and heavy chain respectively containing DNAencoding constant regions of human kappa light chain and human gamma-4heavy chain. The V_(L) region was sub-cloned into the expression vectorby restriction digest and ligation from the sequencing vector, usingSfuI and BsiWI restriction sites, creating plasmid pMRR10(544cL). Theheavy chain DNA was amplified by PCR using a 5′ primer to introduce asignal peptide, since this was not obtained in the cloning strategy—amouse heavy chain antibody leader from a different in-house hybridoma(termed 162) was employed. The 5′ primer had the following sequence:^(5′)GCGCGCAAGCTTGCCGCCACCATGGACTTCGGATTCTCTCTCGTGTTCCTGGCACTCATTCTCAAGGGAGTGCAGTGTGAGGTGCAGCTCGTCGAGTCTGG^(3′) (SEQ ID NO:51).

[0129] The reverse primer was identical to that used in the originalV_(H) gene cloning. The resultant PCR product was digested with enzymesHindIII and ApaI, was sub-cloned, and its DNA sequence was confirmed,creating plasmid pMRR14(544cH). Transient co-transfection of bothexpression vectors into CHO cells generated chimeric c5/44 antibody.This was achieved using the Lipofectamine reagent according to themanufacturer's protocols (InVitrogen:Life Technology, Groningen, TheNetherlands. Catalogue no. 11668-027).

[0130] Removal of Glycosylation Site and Reactive Lysine

[0131] A potential N-linked glycosylation site sequence was observed inCDR-H2, having the amino acid sequence N-Y-T (FIG. 3). SDS-PAGE, Westernblotting and carbohydrate staining of gels of 5/44 and its fragments(including Fab) indicated that this site was indeed glycosylated (notshown). In addition, a lysine residue was observed at an exposedposition within CDR-H2, which had the potential to reduce the bindingaffinity of the antibody by providing an additional site for conjugationwith an agent with which the antibody may be conjugated.

[0132] A PCR strategy was used to introduce amino acid substitutionsinto the CDR-H2 sequence in an attempt to remove the glycosylation siteand/or the reactive lysine, as shown in FIG. 4. Forward primers encodingthe mutations N55Q, T57A or T57V were used to remove the glycosylationsite (FIG. 4) and a fourth forward primer containing the substitutionK60R, was generated to remove the reactive lysine residue (FIG. 4). Aframework 4 reverse primer was used in each of these PCR amplifications.The PCR products were digested with the enzymes XbaI and ApaI and wereinserted into pMRR14(544cH) (also cleaved with XbaI and ApaI) togenerate expression plasmids encoding these mutants. The N55Q, T57A andT57V mutations ablate the glycosylation site by changing the amino acidsequence away from the consensus N-X-T/S whilst the K60R mutationreplaces the potentially reactive lysine with the similarly positivelycharged residue arginine. The resultant cH variant plasmids wereco-transfected with the cL plasmid to generate expressed chimericantibody variants.

[0133] Evaluation of Activities of Chimeric Genes

[0134] The activities of the chimeric genes were evaluated followingtransient transfection into CHO cells.

[0135] c) Determination of Affinity Constants by BiaCore Analysis.

[0136] The affinities of chimeric 5/44 or its variants, which have hadtheir glycosylation site or their reactive lysine removed, wereinvestigated using BIA technology for binding to CD22-mFc constructs.The results are shown in FIG. 8. All binding measurements were performedin the BIAcore™ 2000 instrument (Pharmacia Biosensor AB, Uppsala,Sweden). The assay was performed by capture of CD22mFc via theimmobilised anti-mouse Fc. The antibody was in the soluble phase.Samples, standard, and controls (50 ul) were injected over immobilisedanti-mouse Fc followed by antibody in the soluble phase. After eachcycle the surface was regenerated with 50 ul of 40 mM HCl at 30ul/min.The kinetic analysis was performed using the BIAevaluation 3.1 software(Pharmacia).

[0137] Removal of the glycosylation site in construct T57A resulted in aslightly faster on-rate and a significantly slower off-rate compared tothe chimeric 5/44, giving an affinity improvement of approximately5-fold. The N55Q mutation had no effect on affinity. This result wasunexpected as it suggests that the removal of the carbohydrate itselfapparently has no effect on binding (as with the N55Q change). Theimproved affinity was observed only with the T57A change. One possibleexplanation is that, regardless of the presence of carbohydrate, thethreonine at position 57 exerts a negative effect on binding that isremoved on conversion of threonine to alanine. The hypothesis that thesmall size of alanine is important, and that the negative effect ofthreonine is related to its size, is supported from the result obtainedusing the T57V mutation: that replacement with valine at position 57 isnot beneficial (results not shown).

[0138] Removal of the lysine by the K60R mutation had a neutral effecton affinity, i.e. the introduction of arginine removes a potentialreactive site without compromising affinity.

[0139] The mutations for removal of the glycosylation site and forremoval of the reactive lysine were therefore both included in thehumanisation design.

Example 2: CDR-Grafting of 5/44

[0140] The molecular cloning of genes for the variable regions of theheavy and light chains of the 5/44 antibody and their use to producechimeric (mouse/human) 5/44 antibodies has been described above. Thenucleotide and amino acid sequences of the mouse 5/44 V_(L) and V_(H)domains are shown in FIGS. 2 and 3 (SEQ ID NOS:7 and 8), respectively.This example describes the CDR-grafting of the 5/44 antibody onto humanframeworks to reduce potential immunogenicity in humans, according tothe method of Adair et al., (WO91/09967).

[0141] CDR-Grafting of 5/44 Light Chain

[0142] Protein sequence alignment with consensus sequences from humansub-group I kappa light chain V region indicated 64% sequence identity.Consequently, for constructing the CDR-grafted light chain, the acceptorframework regions chosen corresponded to those of the human VK sub-groupI germline O12,DPK9 sequence. The framework 4 acceptor sequence wasderived from the human J-region germline sequence JK1.

[0143] A comparison of the amino acid sequences of the framework regionsof murine 5/44 and the acceptor sequence is given in FIG. 5 and showsthat there are 27 differences between the donor and acceptor chains. Ateach position, an analysis was made of the potential of the murineresidue to contribute to antigen binding, either directly or indirectly,through effects on packing or at the V_(H)/V_(L) interface. If a murineresidue was considered important and sufficiently different from thehuman residue in terms of size, polarity or charge, then that murineresidue was retained. Based on this analysis, two versions of theCDR-grafted light chain, having the sequences given in SEQ ID NO:19 andSEQ ID NO:20 (FIG. 5), were constructed.

[0144] CDR-Grafting of 5/44 Heavy Chain

[0145] CDR-grafting of 5/44 heavy chain was accomplished using the samestrategy as described for the light chain. The V-domain of 5/44 heavychain was found to be homologous to human heavy chains belonging tosub-group I (70% sequence identity) and therefore the sequence of thehuman sub-group I germline framework VH1-3,DP7 was used as an acceptorframework. The framework 4 acceptor sequences were derived from humanJ-region germline sequence JH4.

[0146] A comparison of 5/44 heavy chain with the framework regions isshown in FIG. 6 where it can be seen that the 5/44 heavy chain differsfrom the acceptor sequence at 22 positions. Analysis of the contributionthat any of these might make to antigen binding led to 5 versions of theCDR-grafted heavy chains being constructed, having the sequences givenin SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26 and SEQ IDNO:27 (FIG. 6).

[0147] Construction of Genes for Grafted Sequences.

[0148] Genes were designed to encode the grafted sequences gH1 and gL1,and a series of overlapping oligonucleotides were designed andconstructed (FIG. 9). A PCR assembly technique was employed to constructthe CDR-grafted V-region genes. Reaction volumes of 100 ul were set upcontaining 10 mM Tris-HCl pH8.3, 1.5 mM MgCl2, 50 mM KCl, 0.001%gelatin, 0.25 mM each deoxyribonucleoside triphosphate, 1 pmole each ofthe ‘internal’ primers (T1, T2, T3, B1, B2, B3), 10 pmole each of the‘external’ primers (F1, R1), and 1 unit of Taq polymerase (AmpliTaq,Applied BioSystems, catalogue no. N808-0171). PCR cycle parameters were94° C. for 1 minute, 55° C. for 1 minute and 72° C. for 1 minute, for 30cycles. The reaction products were then run on a 1.5% agarose gel,excised and recovered using QIAGEN spin columns (QIAquick gel extractionkit, cat no. 28706). The DNA was eluted in a volume of 30 μl. Aliquots(1 μl) of the gH1 and gL1 DNA were then cloned into the InVitrogen TOPOTA cloning vector pCR2.1 TOPO (catalogue no. K4500-01) according to themanufacturer's instructions. This non-expression vector served as acloning intermediate to facilitate sequencing of a large number ofclones. DNA sequencing using vector-specific primers was used toidentify correct clones containing gH1 and gL1, creating plasmids pCR2.1(544gH1) and pCR2.1(544gL1) (FIG. 10).

[0149] An oligonucleotide cassette replacement method was used to createthe humanised grafts gH4,5,6 and 7, and gL2. FIG. 11 shows the design ofthe oligonucleotide cassettes. To construct each variant, the vector(pCR2.1(544gH1) or pCR2.1(544gL1)) was cut with the restriction enzymesshown (XmaI/SacII for the heavy chain, XmaI/BstEII for the light chain).The large vector fragment was gel purified from agarose and was used inligation with the oligonucleotide cassette. These cassettes are composedof 2 complementary oligonucleotides (shown in FIG. 11), mixed at aconcentration of 0.5 pmoles/μl in a volume of 200 μl 12.5 mM Tris-HCl pH7.5, 2.5 mM MgCl₂, 25 mM NaCl, 0.25 mM dithioerythritol. Annealing wasachieved by heating to 95° C. for 3 minutes in a waterbath (volume 500ml) then allowing the reaction to slow-cool to room temperature. Theannealed oligonucleotide cassette was then diluted ten-fold in waterbefore ligation into the appropriately cut vector. DNA sequencing wasused to confirm the correct sequence, creating plasmids pCR2.1(5/44-gH4-7) and pCR2.1(5/44-gL2). The verified grafted sequences werethen sub-cloned into the expression vectors pMRR14 (heavy chain) andpMR10.1 (light chain).

[0150] CD22 Binding Activity of CDR-grafted Sequences

[0151] The vectors encoding grafted variants were co-transfected intoCHO cells in a variety of combinations, together with the originalchimeric antibody chains. Binding activity was compared in a competitionassay, competing the binding of the original mouse 5/44 antibody forbinding to Ramos cells (obtained from ATCC, a Burkitt's lymphomalymphoblast human cell line expressing surface CD22). This assay wasconsidered the best way to compare grafts in their ability to bind tocell surface CD22. The results are shown in FIG. 8. As can be seen,there is very little difference between any of the grafts, allperforming more effectively than the chimeric at competing against themurine parent. The introduction of the 3 additional human residues atthe end of CDR-H3 (gH5 and gH7) does not appear to have affectedbinding.

[0152] The graft combination with the least number of murine residueswas selected, gL1gH7. The light chain graft gL1 has 6 donor residues.Residues V2, V4, L37 and Q45 are potentially important packing residues.Residue H38 is at the V_(H)/V_(L) interface. Residue D60 is a surfaceresidue close to the CDR-L2 and may directly contribute to antigenbinding. Of these residues, V2, L37, Q45 and D60 are found in germlinesequences of human kappa genes from other sub-groups. The heavy chaingraft gH7 has 4 donor framework residues (Residue R28 is considered tobe part of CDR-H1 under the structural definition used in CDR-grafting(se Adair et al (1991 WO91/09967)). Residues E1 and A71 are surfaceresidues close to the CDR's. Residue 148 is a potential packing residue.Residue T93 is present at the V_(H)/V_(L) interface. Of these residues,E1 and A71 are found in other germline genes of human sub-group I.Residue 148 is found in human germline sub-group 4, and T73 is found inhuman germline sub-group 3.

[0153] The full DNA and protein sequence of both the light chain andheavy chain, including approximate position of introns within theconstant region genes provided by the vectors, are shown in FIG. 13 andare given in SEQ ID NO:29 and SEQ ID NO:28 respectively for the lightchain and SEQ ID NO: 31 and SEQ ID NO:30 respectively for the heavychain.

[0154] DNA encoding these light and heavy chain genes was excised fromthese vectors. Heavy chain DNA was digested at the 5′ HindIII site, thenwas treated with the Klenow fragment of E. coli DNA polymerase I tocreate a 5′ blunt end. Cleavage at the 3′ EcoRI site resulted in theheavy chain fragment which was purified from agarose gels. In the sameway, a light chain frament was produced, blunted at the 5′ SfuI site andwith a 3′ EcoRI site. Both fragments were cloned into DHFR basedexpression vectors and used to generate stable cell lines in CHO cells.

[0155] All references and patents cited herein are hereby incorporatedby reference in their entireties.

1 51 1 5 PRT mouse DOMAIN mouse monoclonal 5/44 CDR-H1 1 Asn Tyr Trp IleHis 1 5 2 17 PRT mouse DOMAIN mouse monoclonal 5/44 CDR-H2 2 Gly Ile AsnPro Gly Asn Asn Tyr Thr Thr Tyr Lys Arg Asn Leu Lys 1 5 10 15 Gly 3 12PRT mouse DOMAIN mouse monoclonal 5/44 CDR-H3 3 Glu Gly Tyr Gly Asn TyrGly Ala Trp Phe Ala Tyr 1 5 10 4 16 PRT mouse DOMAIN mouse monoclonal5/44 CDR-L1 4 Arg Ser Ser Gln Ser Leu Ala Asn Ser Tyr Gly Asn Thr PheLeu Ser 1 5 10 15 5 7 PRT mouse DOMAIN mouse monoclonal 5/44 CDR-L2 5Gly Ile Ser Asn Arg Phe Ser 1 5 6 9 PRT mouse DOMAIN mouse monoclonal5/44 CDR-L3 6 Leu Gln Gly Thr His Gln Pro Tyr Thr 1 5 7 113 PRT mouseDOMAIN mouse monoclonal 5/44 VL domain 7 Asp Val Val Val Thr Gln Thr ProLeu Ser Leu Pro Val Ser Phe Gly 1 5 10 15 Asp Gln Val Ser Ile Ser CysArg Ser Ser Gln Ser Leu Ala Asn Ser 20 25 30 Tyr Gly Asn Thr Phe Leu SerTrp Tyr Leu His Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr GlyIle Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Thr Gly Ser GlySer Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Thr Ile Lys Pro GluAsp Leu Gly Met Tyr Tyr Cys Leu Gln Gly 85 90 95 Thr His Gln Pro Tyr ThrPhe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 Arg 8 121 PRT mouseDOMAIN mouse monoclonal 5/44 VH domain 8 Glu Val Gln Leu Gln Gln Ser GlyThr Val Leu Ala Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys LysAla Ser Gly Tyr Arg Phe Thr Asn Tyr 20 25 30 Trp Ile His Trp Val Lys GlnArg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Gly Ile Asn Pro Gly AsnAsn Tyr Thr Thr Tyr Lys Arg Asn Leu 50 55 60 Lys Gly Lys Ala Thr Leu ThrAla Val Thr Ser Ala Ser Thr Ala Tyr 65 70 75 80 Met Asp Leu Ser Ser LeuThr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Glu Gly Tyr GlyAsn Tyr Gly Ala Trp Phe Ala Tyr Trp Gly 100 105 110 Gln Gly Thr Leu ValThr Val Ser Ser 115 120 9 13 PRT Artificial Sequence DOMAIN cH 9 Gly AsnAsn Tyr Thr Thr Tyr Lys Arg Asn Leu Lys Gly 1 5 10 10 13 PRT ArtificialSequence DOMAIN CDR-H2 MUTATION N55Q 10 Gly Asn Gln Tyr Thr Thr Tyr LysArg Asn Leu Lys Gly 1 5 10 11 13 PRT Artificial Sequence DOMAIN CDR-H2MUTATION T57A 11 Gly Asn Asn Tyr Ala Thr Tyr Lys Arg Asn Leu Lys Gly 1 510 12 13 PRT Artificial Sequence DOMAIN CDR-H2 MUTATION T57V 12 Gly AsnAsn Tyr Val Thr Tyr Lys Arg Asn Leu Lys Gly 1 5 10 13 17 PRT ARTIFICIALSEQUENCE DOMAIN CDR-H2 MUTATION (T57A) H′ 13 Gly Ile Asn Pro Gly Asn AsnTyr Ala Thr Tyr Lys Arg Asn Leu Lys 1 5 10 15 Gly 14 13 PRT ArtificialSequence DOMAIN CDR-H2 MUTATION K60R 14 Gly Asn Asn Tyr Thr Thr Tyr ArgArg Asn Leu Lys Gly 1 5 10 15 17 PRT ARTIFICIAL SEQUENCE DOMAIN CDR-H2MUTATION (K60R) H′′ 15 Gly Ile Asn Pro Gly Asn Asn Tyr Thr Thr Tyr ArgArg Asn Leu Lys 1 5 10 15 Gly 16 17 PRT ARTIFICIAL SEQUENCE DOMAINCDR-H2 MUTATION (T57A K60R) H′′′ 16 Gly Ile Asn Pro Gly Asn Asn Tyr AlaThr Tyr Arg Arg Asn Leu Lys 1 5 10 15 Gly 17 70 PRT Homo sapiens DOMAINDPK9 HUMAN ACCEPTOR FRAMEWORK 17 Asp Ile Gln Met Thr Gln Ser Pro Ser SerLeu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Trp TyrGln Gln Lys Pro Gly Lys Ala 20 25 30 Pro Lys Leu Leu Ile Tyr Gly Val ProSer Arg Phe Ser Gly Ser Gly 35 40 45 Ser Gly Thr Asp Phe Thr Leu Thr IleSer Ser Leu Gln Pro Glu Asp 50 55 60 Phe Ala Thr Tyr Tyr Cys 65 70 18 11PRT Homo sapiens DOMAIN JK1 HUMAN ACCEPTOR FRAMEWORK 18 Phe Gly Gln GlyThr Lys Val Glu Ile Lys Arg 1 5 10 19 113 PRT Artificial Sequence CHAINgL1 19 Asp Val Gln Val Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 15 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ser Ser Gln Ser Leu Ala Asn Ser20 25 30 Tyr Gly Asn Thr Phe Leu Ser Trp Tyr Leu His Lys Pro Gly Lys Ala35 40 45 Pro Gln Leu Leu Ile Tyr Gly Ile Ser Asn Arg Phe Ser Gly Val Pro50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile65 70 75 80 Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Leu GlnGly 85 90 95 Thr His Gln Pro Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu IleLys 100 105 110 Arg 20 113 PRT Artificial Sequence CHAIN gL2 20 Asp ValVal Val Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 AspArg Val Thr Ile Thr Cys Arg Ser Ser Gln Ser Leu Ala Asn Ser 20 25 30 TyrGly Asn Thr Phe Leu Ser Trp Tyr Leu His Lys Pro Gly Lys Ala 35 40 45 ProGln Leu Leu Ile Tyr Gly Ile Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 AspArg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile 65 70 75 80Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Gly 85 90 95Thr His Gln Pro Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105110 Arg 21 80 PRT Homo sapiens CHAIN DP7 HUMAN ACCEPTOR FRAMEWORK 21 GlnVal Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Trp Val 20 25 30Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly Lys Phe Gln Gly 35 40 45Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr Met Glu 50 55 60Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg 65 70 7580 22 11 PRT Homo sapiens DOMAIN JH4 HUMAN ACCEPTOR FRAMEWORK 22 Trp GlyGln Gly Thr Leu Val Thr Val Ser Ser 1 5 10 23 121 PRT ArtificialSequence CHAIN gH1 23 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val LysLys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly TyrArg Phe Thr Asn Tyr 20 25 30 Trp Ile His Trp Val Arg Gln Ala Pro Gly GlnGly Leu Glu Trp Ile 35 40 45 Gly Gly Ile Asn Pro Gly Asn Gln Tyr Thr ThrTyr Lys Arg Asn Leu 50 55 60 Lys Gly Arg Ala Thr Leu Thr Ala Asp Thr SerThr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu AspThr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Glu Gly Tyr Gly Asn Tyr Gly AlaTrp Phe Ala Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser115 120 24 121 PRT Artificial Sequence CHAIN gH4 24 Glu Val Gln Leu ValGln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys ValSer Cys Lys Ala Ser Gly Tyr Arg Phe Thr Asn Tyr 20 25 30 Trp Ile His TrpVal Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Gly Ile AsnPro Gly Asn Asn Tyr Ala Thr Tyr Arg Arg Asn Leu 50 55 60 Lys Gly Arg AlaThr Leu Thr Ala Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu LeuSer Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg GluGly Tyr Gly Asn Tyr Gly Ala Trp Phe Ala Tyr Trp Gly 100 105 110 Gln GlyThr Leu Val Thr Val Ser Ser 115 120 25 121 PRT Artificial Sequence CHAINgH5 25 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 15 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Arg Phe Thr Asn Tyr20 25 30 Trp Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile35 40 45 Gly Gly Ile Asn Pro Gly Asn Asn Tyr Ala Thr Tyr Arg Arg Asn Leu50 55 60 Lys Gly Arg Val Thr Met Thr Ala Asp Thr Ser Thr Ser Thr Val Tyr65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr TyrCys 85 90 95 Thr Arg Glu Gly Tyr Gly Asn Tyr Gly Ala Trp Phe Ala Tyr TrpGly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 26 121 PRTArtificial Sequence CHAIN gH6 26 Glu Val Gln Leu Val Gln Ser Gly Ala GluVal Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala SerGly Tyr Arg Phe Thr Asn Tyr 20 25 30 Trp Ile His Trp Val Arg Gln Ala ProGly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Gly Ile Asn Pro Gly Asn Asn TyrAla Thr Tyr Arg Arg Lys Phe 50 55 60 Gln Gly Arg Ala Thr Leu Thr Ala AspThr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg SerGlu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Glu Gly Tyr Gly Asn TyrGly Ala Trp Phe Ala Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr ValSer Ser 115 120 27 121 PRT Artificial Sequence CHAIN gH7 27 Glu Val GlnLeu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser ValLys Val Ser Cys Lys Ala Ser Gly Tyr Arg Phe Thr Asn Tyr 20 25 30 Trp IleHis Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly GlyIle Asn Pro Gly Asn Asn Tyr Ala Thr Tyr Arg Arg Lys Phe 50 55 60 Gln GlyArg Val Thr Met Thr Ala Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 MetGlu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 ThrArg Glu Gly Tyr Gly Asn Tyr Gly Ala Trp Phe Ala Tyr Trp Gly 100 105 110Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 28 239 PRT ArtificialSequence CHAIN Full sequence of grafted light chain 28 Met Lys Leu ProVal Arg Leu Leu Val Leu Leu Leu Phe Trp Ile Pro 1 5 10 15 Ala Ser ArgGly Asp Val Gln Val Thr Gln Ser Pro Ser Ser Leu Ser 20 25 30 Ala Ser ValGly Asp Arg Val Thr Ile Thr Cys Arg Ser Ser Gln Ser 35 40 45 Leu Ala AsnSer Tyr Gly Asn Thr Phe Leu Ser Trp Tyr Leu His Lys 50 55 60 Pro Gly LysAla Pro Gln Leu Leu Ile Tyr Gly Ile Ser Asn Arg Phe 65 70 75 80 Ser GlyVal Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe 85 90 95 Thr LeuThr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr 100 105 110 CysLeu Gln Gly Thr His Gln Pro Tyr Thr Phe Gly Gln Gly Thr Lys 115 120 125Val Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro 130 135140 Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu 145150 155 160 Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys ValAsp 165 170 175 Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr GluGln Asp 180 185 190 Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu ThrLeu Ser Lys 195 200 205 Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys GluVal Thr His Gln 210 215 220 Gly Leu Ser Ser Pro Val Thr Lys Ser Phe AsnArg Gly Glu Cys 225 230 235 29 781 DNA ARTIFICIAL CDS Full DNA sequenceof grafted light chain 29 ttcgaagccg ccaccatgaa gttgcctgtt aggctgttggtgcttctgtt gttctggatt 60 cctgcttccc ggggtgacgt tcaagtgacc cagagcccatccagcctgag cgcatctgta 120 ggagaccggg tcaccatcac ttgtagatcc agtcagagtcttgcaaacag ttatgggaac 180 acctttttgt cttggtatct gcacaaacca ggtaaagccccacaattgct catctacgga 240 atctctaaca gatttagtgg tgtaccagac aggttcagcggttccggaag tggtactgat 300 ttcaccctca cgatctcgtc tctccagcca gaagatttcgccacttatta ctgtttacaa 360 ggtacacatc agccgtacac attcggtcag ggtactaaagtagaaatcaa acgtacggta 420 gcggccccat ctgtcttcat cttcccgcca tctgatgagcagttgaaatc tggaactgcc 480 tctgttgtgt gcctgctgaa taacttctat cccagagaggccaaagtaca gtggaaggtg 540 gataacgccc tccaatcggg taactcccag gagagtgtcacagagcagga cagcaaggac 600 agcacctaca gcctcagcag caccctgacg ctgagcaaagcagactacga gaaacacaaa 660 gtctacgcct gcgaagtcac ccatcagggc ctgagctcgcccgtcacaaa gagcttcaac 720 aggggagagt gttagaggga gaagtgcccc cacctgctcctcagttccag cctgggaatt 780 c 781 30 467 PRT Artificial Sequence CHAINFull sequence of grafted heavy chain 30 Met Asp Phe Gly Phe Ser Leu ValPhe Leu Ala Leu Ile Leu Lys Gly 1 5 10 15 Val Gln Cys Glu Val Gln LeuVal Gln Ser Gly Ala Glu Val Lys Lys 20 25 30 Pro Gly Ala Ser Val Lys ValSer Cys Lys Ala Ser Gly Tyr Arg Phe 35 40 45 Thr Asn Tyr Trp Ile His TrpVal Arg Gln Ala Pro Gly Gln Gly Leu 50 55 60 Glu Trp Ile Gly Gly Ile AsnPro Gly Asn Asn Tyr Ala Thr Tyr Arg 65 70 75 80 Arg Lys Phe Gln Gly ArgVal Thr Met Thr Ala Asp Thr Ser Thr Ser 85 90 95 Thr Val Tyr Met Glu LeuSer Ser Leu Arg Ser Glu Asp Thr Ala Val 100 105 110 Tyr Tyr Cys Thr ArgGlu Gly Tyr Gly Asn Tyr Gly Ala Trp Phe Ala 115 120 125 Tyr Trp Gly GlnGly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys 130 135 140 Gly Pro SerVal Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu 145 150 155 160 SerThr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro 165 170 175Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr 180 185190 Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val 195200 205 Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn210 215 220 Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val GluSer 225 230 235 240 Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro GluPhe Leu Gly 245 250 255 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro LysAsp Thr Leu Met 260 265 270 Ile Ser Arg Thr Pro Glu Val Thr Cys Val ValVal Asp Val Ser Gln 275 280 285 Glu Asp Pro Glu Val Gln Phe Asn Trp TyrVal Asp Gly Val Glu Val 290 295 300 His Asn Ala Lys Thr Lys Pro Arg GluGlu Gln Phe Asn Ser Thr Tyr 305 310 315 320 Arg Val Val Ser Val Leu ThrVal Leu His Gln Asp Trp Leu Asn Gly 325 330 335 Lys Glu Tyr Lys Cys LysVal Ser Asn Lys Gly Leu Pro Ser Ser Ile 340 345 350 Glu Lys Thr Ile SerLys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 355 360 365 Tyr Thr Leu ProPro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser 370 375 380 Leu Thr CysLeu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 385 390 395 400 TrpGlu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 405 410 415Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val 420 425430 Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met 435440 445 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser450 455 460 Leu Gly Lys 465 31 2160 DNA Artificial Sequence CDS Full DNAsequence of grafted heavy chain 31 aagcttgccg ccaccatgga cttcggattctctctcgtgt tcctggcact cattctcaag 60 ggagtgcagt gtgaggtgca attggtccagtcaggagcag aggttaagaa gcctggtgct 120 tccgtcaaag tttcgtgtaa ggctagcggctacaggttca caaattattg gattcattgg 180 gtcaggcagg ctccgggaca aggcctggaatggatcggtg gcattaatcc cgggaataac 240 tacgctacat ataggagaaa attccagggcagagttacga tgaccgcgga cacctccaca 300 agcactgtct acatggagct gtcatctctgagatccgagg acaccgcagt gtactattgt 360 actagagaag gctacggtaa ttacggagcctggttcgcct actggggcca gggtacccta 420 gtcacagtct cctcagcttc tacaaagggcccatccgtct tccccctggc gccctgctcc 480 aggagcacct ccgagagcac agccgccctgggctgcctgg tcaaggacta cttccccgaa 540 ccggtgacgg tgtcgtggaa ctcaggcgccctgaccagcg gcgtgcacac cttcccggct 600 gtcctacagt cctcaggact ctactccctcagcagcgtgg tgaccgtgcc ctccagcagc 660 ttgggcacga agacctacac ctgcaacgtagatcacaagc ccagcaacac caaggtggac 720 aagagagttg gtgagaggcc agcacagggagggagggtgt ctgctggaag ccaggctcag 780 ccctcctgcc tggacgcacc ccggctgtgcagccccagcc cagggcagca aggcatgccc 840 catctgtctc ctcacccgga ggcctctgaccaccccactc atgcccaggg agagggtctt 900 ctggattttt ccaccaggct ccgggcagccacaggctgga tgcccctacc ccaggccctg 960 cgcatacagg ggcaggtgct gcgctcagacctgccaagag ccatatccgg gaggaccctg 1020 cccctgacct aagcccaccc caaaggccaaactctccact ccctcagctc agacaccttc 1080 tctcctccca gatctgagta actcccaatcttctctctgc agagtccaaa tatggtcccc 1140 catgcccacc atgcccaggt aagccaacccaggcctcgcc ctccagctca aggcgggaca 1200 ggtgccctag agtagcctgc atccagggacaggccccagc cgggtgctga cgcatccacc 1260 tccatctctt cctcagcacc tgagttcctggggggaccat cagtcttcct gttcccccca 1320 aaacccaagg acactctcat gatctcccggacccctgagg tcacgtgcgt ggtggtggac 1380 gtgagccagg aagaccccga ggtccagttcaactggtacg tggatggcgt ggaggtgcat 1440 aatgccaaga caaagccgcg ggaggagcagttcaacagca cgtaccgtgt ggtcagcgtc 1500 ctcaccgtcc tgcaccagga ctggctgaacggcaaggagt acaagtgcaa ggtctccaac 1560 aaaggcctcc cgtcctccat cgagaaaaccatctccaaag ccaaaggtgg gacccacggg 1620 gtgcgagggc cacatggaca gaggtcagctcggcccaccc tctgccctgg gagtgaccgc 1680 tgtgccaacc tctgtcccta cagggcagccccgagagcca caggtgtaca ccctgccccc 1740 atcccaggag gagatgacca agaaccaggtcagcctgacc tgcctggtca aaggcttcta 1800 ccccagcgac atcgccgtgg agtgggagagcaatgggcag ccggagaaca actacaagac 1860 cacgcctccc gtgctggact ccgacggctccttcttcctc tacagcaggc taaccgtgga 1920 caagagcagg tggcaggagg ggaatgtcttctcatgctcc gtgatgcatg aggctctgca 1980 caaccactac acacagaaga gcctctccctgtctctgggt aaatgagtgc cagggccggc 2040 aagcccccgc tccccgggct ctcggggtcgcgcgaggatg cttggcacgt accccgtcta 2100 catacttccc aggcacccag catggaaataaagcacccac cactgccctg gctcgaattc 2160 32 94 DNA Artificial Sequence CDS544gH1 T1 32 agtgtgaggt gcaattggtc cagtcaggag cagaggttaa gaagcctggtgcttccgtca 60 aagtttcgtg taaggctagc ggctacaggt tcac 94 33 96 DNAArtificial Sequence CDS 544gH1 T2 33 gtggcattaa tcccgggaat cagtacactacatataaaag aaatctaaag ggcagagcaa 60 cgctgaccgc ggacacctcc acaagcactgtctaca 96 34 95 DNA Artificial Sequence CDS 544gH1 T3 34 agagaaggctacggtaatta cggagcctgg ttcgcctact ggggccaggg taccctagtc 60 acagtctcctcagcttctac aaagggccca agaaa 95 35 94 DNA Artificial Sequence CDS 544 gH1B1 35 ggaccaattg cacctcacac tgcactccct tgagaatgag tgccaggaac acgagagaga60 atccgaagtc catggtggcg gcaagctttt attc 94 36 97 DNA ArtificialSequence CDS 544gH1 B2 36 gattcccggg attaatgcca ccgatccatt ccaggccttgtcccggagcc tgcctgaccc 60 aatgaatcca ataatttgtg aacctgtagc cgctagc 97 3793 DNA Artificial Sequence CDS 544gH1 B3 37 cgtaattacc gtagccttctctagtacaat agtacactgc ggtgtcctcg gatctcagag 60 atgacagctc catgtagacagtgcttgtgg agg 93 38 21 DNA Artificial Sequence CDS 544gH1 F1 38gaataaaagc ttgccgccac c 21 39 22 DNA Artificial Sequence CDS 544gH1 R139 tttcttgggc cctttgtaga ag 22 40 87 DNA Artificial Sequence CDS 544 gL1T1 40 gcttcccggg gtgacgttca agtgacccag agcccatcca gcctgagcgc atctgtagga60 gaccgggtca ccatcacttg tagatcc 87 41 90 DNA Artificial Sequence CDS544 gL1 T2 41 tatctgcaca aaccaggtaa agccccacaa ttgctcatct acggaatctctaacagattt 60 agtggtgtac cagacaggtt cagcggttcc 90 42 91 DNA ArtificialSequence CDS 544gL1 T3 42 agatttcgcc acttattact gtttacaagg tacacatcagccgtacacat tcggtcaggg 60 tactaaagta gaaatcaaac gtacggcgtg c 91 43 88 DNAARTIFICIAL SEQUENCE CDS 544gL1 B1 43 gaacgtcacc ccgggaagca ggaatccagaacaacagaag caccaacagc ctaacaggca 60 acttcatggt ggcggcttcg aatcatcc 88 4488 DNA Artificial Sequence CDS 544gL1 B2 44 ctttacctgg tttgtgcagataccaagaca aaaaggtgtt cccataactg tttgcaagac 60 tctgactgga tctacaagtgatggtgac 88 45 90 DNA Artificial Sequence CDS 544gL1 B3 45 aacagtaataagtggcgaaa tcttctggct ggagagacga gatcgtgagg gtgaaatcag 60 taccacttccggaaccgctg aacctgtctg 90 46 20 DNA Artificial Sequence CDS 544gL1 F1 46ggatgattcg aagccgccac 20 47 21 DNA ARTIFICIAL SEQUENCE CDS 544gL1 R1 47gcacgccgta cgtttgattt c 21 48 339 DNA mouse CDS DNA sequence of mousemonoclonal 5/44 VL 48 gatgttgtgg tgactcaaac tccactctcc ctgcctgtcagctttggaga tcaagtttct 60 atctcttgca ggtctagtca gagtcttgca aacagttatgggaacacctt tttgtcttgg 120 tacctgcaca agcctggcca gtctccacag ctcctcatctatgggatttc caacagattt 180 tctggggtgc cagacaggtt cactggcagt ggttcagggacagatttcac actcaagatc 240 agcacaataa agcctgagga cttgggaatg tattactgcttacaaggtac acatcagccg 300 tacacgttcg gaggggggac caagctggaa ataaaacgt 33949 363 DNA mouse CDS DNA sequence of mouse monoclonal 5/44 VH 49gaggtccaac tgcagcagtc tgggactgta ctggcaaggc ctggggcttc cgtgaagatg 60tcctgcaagg cttctggcta caggtttacc aactactgga ttcactgggt aaaacagagg 120cctgggcagg gtctagaatg gattggtggt attaatcctg gaaataatta tactacgtat 180aagaggaact tgaagggcaa ggccacactg actgcagtca catccgccag cactgcctac 240atggacctca gcagcctgac aagtgaggac tctgcggtct attactgtac aagagagggc 300tatggtaact acggggcctg gtttgcttac tggggccagg ggactctggt caccgtctcc 360tca 363 50 9 DNA Artificial Sequence PRIMER sequence withinoligonucleotide primer 50 gccgccacc 9 51 101 DNA Artificial SequencePRIMER 5′ oligonucleotide primer 51 gcgcgcaagc ttgccgccac catggacttcggattctctc tcgtgttcct ggcactcatt 60 ctcaagggag tgcagtgtga ggtgcagctcgtcgagtctg g 101

What is claimed is:
 1. An antibody molecule having specificity for humanCD22, comprising a heavy chain wherein the variable domain comprises aCDR having at least one of the sequences given as H1 in FIG. 1 (SEQ IDNO:1) for CDR-H1, as H2 in FIG. 1 (SEQ ID NO:2) or H2′ (SEQ ID NO:13) orH2″ (SEQ ID NO:15) or H2′″ (SEQ ID NO:16) for CDR-H2 or as H3 in FIG. 1(SEQ ID NO:3) for CDR-H3.
 2. An antibody molecule having specificity forhuman CD22, comprising a light chain wherein the variable domaincomprises a CDR having at least one of the sequences given as L1 in FIG.1 (SEQ ID NO:4) for CDR-L1, as L2 in FIG. 1 (SEQ ID NO:5) for CDR-L2 oras L3 in FIG. 1 (SEQ ID NO:6) for CDR-L3.
 3. The antibody molecule ofclaim 1 or claim 2 comprising a heavy chain wherein the variable domaincomprises a CDR having at least one of the sequences given in SEQ IDNO:1 for CDR-H1, SEQ ID NO:2 or SEQ ID NO:13 or SEQ ID NO:15 or SEQ IDNO:16 for CDR-H2 or SEQ ID NO:3 for CDR-H3 and a light chain wherein thevariable domain comprises a CDR having at least one of the sequencesgiven in SEQ ID NO:4 for CDR-L1, SEQ ID NO:5 for CDR-L2 or SEQ ID NO:6for CDR-L3.
 4. The antibody molecule of claim 3, which comprises SEQ IDNO:1 for CDR-H1, SEQ ID NO: 2 or SEQ ID NO:13 or SEQ ID NO:15 or SEQ IDNO:16 for CDR-H2, SEQ ID NO:3 for CDR-H3, SEQ ID NO:4 for CDR-L1, SEQ IDNO:5 for CDR-L2 and SEQ ID NO:6 for CDR-L3.
 5. The antibody molecule ofany one of claims 1 to 4, which is a CDR-grafted antibody molecule. 6.The antibody molecule of claim 5, wherein the variable domain compriseshuman acceptor framework regions and non-human donor CDRs.
 7. Theantibody molecule of claim 6, wherein the human acceptor frameworkregions of the variable domain of the heavy chain are based on a humansub-group I consensus sequence and comprise donor residues at positions1, 28, 48, 71 and 93 that correspond to the residues at those positionsin SEQ ID NO:8.
 8. The antibody molecule of claim 7, additionallycomprising donor residues at positions 67 and 69 that correspond to theresidues at those positions in SEQ ID NO:8.
 9. The antibody molecule ofany one of claims 6 to 8, wherein the human acceptor framework regionsof the variable domain of the light chain are based on a human sub-groupI consensus sequence and comprise donor residues at positions 2, 4, 37,38, 45 and 60 that correspond to the residues at those positions in SEQID NO:7.
 10. The antibody molecule of claim 9, additionally comprising adonor residue at position 3 that corresponds to the residue at thatposition in SEQ ID NO:7.
 11. An antibody molecule having specificity forhuman CD22, comprising a heavy chain according to either claim 7 orclaim 8, and a light chain according to either claim 9 or claim
 10. 12.The antibody molecule of any one of claims 1 to 11, comprising the lightchain variable region 5/44-gL1 (SEQ ID NO: 19) and the heavy chainvariable region 5/44-gH7 (SEQ ID NO:27).
 13. An antibody molecule havingspecificity for human CD22, comprising a light chain, wherein thesequence of the light chain comprises the sequence given in SEQ IDNO:28.
 14. An antibody molecule having specificity for human CD22,comprising a light chain, wherein the sequence of the light chainconsists of the sequence given in SEQ ID NO:28.
 15. An antibody moleculehaving specificity for human CD22, comprising a heavy chain, wherein thesequence of the heavy chain comprises the sequence given in SEQ IDNO:30.
 16. An antibody molecule having specificity for human CD22,comprising a heavy chain, wherein the sequence of the heavy chainconsists of the sequence given in SEQ ID NO:30.
 17. An antibody moleculehaving specificity for human CD22, having a light chain comprising thesequence given in SEQ ID NO:28 and a heavy chain comprising the sequencegiven in SEQ ID NO:30.
 18. An antibody molecule having specificity forhuman CD22, having a light chain consisting of the sequence given in SEQID NO:28 and a heavy chain consisting of the sequence given in SEQ IDNO:30.
 19. A variant of the antibody molecule of any one of claims 1 to18, which has an improved affinity for CD22.
 20. The variant of claim 19which is obtained by an affinity maturation protocol.
 21. The antibodyof any one of claims 1 to 4, which is murine anti-CD22 monoclonalantibody 5/44, wherein the variable domain of the light chain has thesequence given in SEQ ID NO: 7 and the variable domain of the heavychain has the sequence given in SEQ ID NO:
 8. 22. The antibody moleculeof any one of claims 1 to 4, which is a chimeric antibody moleculecomprising the sequences of the light and heavy chain variable domainsof the monoclonal antibody of claim 21, recited in SEQ ID NO:7 and SEQID NO:8 respectively.
 23. An antibody molecule comprising a hybrid CDRcomprising a truncated donor CDR sequence wherein the missing portion ofthe donor CDR is replaced by a different sequence and forms a functionalCDR.
 24. The antibody molecule of claim 23, wherein the missing part ofthe CDR sequence is from the antibody from which the framework regionsof the antibody molecule are derived.
 25. The antibody molecule of claim24, wherein the missing part of the CDR sequence is derived from agermline antibody having consensus framework regions.
 26. The antibodymolecule of any one of claims 23 to 25, wherein CDR-H2 of the heavychain is hybrid in the antibody molecule.
 27. The antibody molecule ofany one of claims 23 to 26, wherein the truncation of the donor CDR isfrom 1 to 8 amino acids.
 28. The antibody molecule of claim 27, whereinthe truncation is from 4 to 6 amino acids.
 29. The antibody molecule ofany one of claims 23 to 28, wherein the truncation is made at theC-terminus of the CDR.
 30. A DNA sequence encoding the heavy chainand/or the light chain of an antibody molecule according to any one ofclaims 1 to
 29. 31. A cloning or expression vector comprising a DNAsequence according to claim
 30. 32. A host cell comprising a cloning orexpression vector according to claim
 31. 33. An antibody moleculeaccording to any one of claims 1 to 29 or a DNA sequence according toclaim 30 for use in therapy.
 34. The antibody molecule of any one ofclaims 1 to 29 or 33, having specificity for human CD22, or a DNAsequence according to claim 30 for use in treating a pathology mediatedby cells expressing CD22.
 35. An antibody molecule according to claim 33or claim 34 or a DNA sequence according to claim 33 or claim 34, for usein treating malignant lymphoma.
 36. The antibody molecule or DNAsequence of claim 35, wherein the malignant lymphoma is Non-Hodgkin'slymphoma.
 37. Use of the antibody molecule of any one of claims 1 to 29,having specificity for human CD22, or use of a DNA sequence according toclaim 30 in the manufacture of a medicament for the treatment of apathology mediated by cells expressing CD22.
 38. The use of claim 37,wherein the pathology is malignant lymphoma.
 39. Claim 38, wherein themalignant lymphoma is Non-Hodgkin's lymphoma.
 40. A therapeutic ordiagnostic composition comprising the antibody molecule of any one ofclaims 1 to 29 or the DNA sequence of claim
 30. 41. A therapeutic ordiagnostic composition according to claim 40, comprising apharmaceutically acceptable excipient, diluent or carrier.
 42. Atherapeutic or diagnostic composition according to claim 40 or claim 41,additionally comprising anti-T cell, anti-IFNγ or anti-LPS antibodies,or non-antibody ingredients such as xanthines.
 43. A process for theproduction of an antibody molecule according to any one of claims 1 to29 comprising culturing a host cell according to claim 32 underconditions suitable for leading to expression of protein from DNAencoding said antibody molecule and isolating said antibody molecule.44. A process for the preparation of a therapeutic or diagnosticcomposition according to any one of claims 40 to 42, comprising admixingan antibody molecule according to any one of claims 1 to 29 togetherwith a pharmaceutically acceptable excipient, diluent or carrier.
 45. Apolypeptide having the amino acid sequence given in any one of SEQ IDNOs:1 to 28 or SEQ ID NO:30.